Protein interactions with other molecules is basic to biochemistry. Protein interactions include receptor-ligand interactions, antibody-antigen interactions, cell-cell contact and pathogen interactions with target tissues. Protein interactions can involve contact with other proteins, with carbohydrates, oligosaccharides, lipids, metal ions and the like materials.
The basic unit of protein interaction is the region of the protein involved in contact and recognition, and is referred to as the binding site.
There is an increasing need to find new molecules which can effectively modulate a wide range of biological processes, for applications in medicine and agriculture. Thus, there is a need for systematic and rapid development of binding sites on proteins for use in the construction of protein binding site analogs and antagonists, proteins with improved or altered binding specificities and the attendant altered function associated with the altered specificity, and antibodies with unique antigen specificities.
Numerous strategies have been developed for preparing proteins having new binding specificities besides the conventional technique of random screening of natural products. These approaches generally involve the synthetic production of large numbers of random molecules followed by some selection procedure to identify the molecule of interest. For example, epitope libraries have been developed using random polypeptides displayed on the surface of filamentous phage particles. The library is made by synthesizing a repertoire of random oligonucleotides to generate all combinations, followed by their insertion into a phage vector. Each of the sequences is separately cloned and expressed in phage, and the relevant expressed peptide can be selected by finding those phage that bind to the particular target. The phages recovered in this way can be amplified and the selection repeated. The sequence of the peptide is decoded by sequencing the DNA. See for example Cwirla et al., Proc. Natl. Acad. Sci., USA, 87:6378-6382 (1990); Scott et al., Science, 249:386-390 (1990); and Devlin et al., Science, 249:404-406 (1990).
Another approach involves large arrays of peptides that are synthesized in parallel and screened with acceptor molecules labelled with fluorescent or other reporter groups. The sequence of any effective peptide can be decoded from its address in the array. See for example Geysen et al., Proc. Natl. Acad. Sci., USA, 81:3998-4002 (1984); Maeji et al., J. Immunol. Met., 146:83-90 (1992); and Fodor et al., Science, 251: 767-775 (1991).
In another approach, Lam et al., Nature, 354:82-84 (1991) describes combinatorial libraries of peptides that are synthesized on resin beads such that each resin bead contains about 20 pmoles of the same peptide. The beads are screened with labelled acceptor molecules and those with bound acceptor are searched for by visual inspection, physically removed, and the peptide identified by direct sequence analysis. In principle, this method could be used with other chemical entities but it requires sensitive methods for sequence determination.
A different method of solving the problem of identification in a combinatorial peptide library is used by Houghten et al., Nature, 354:84-86 (1991). For hexapeptides of the 20 natural amino acids, 400 separate libraries are synthesized, each with the first two amino acids fixed and the remaining four positions occupied by all possible combinations. An assay, based on competition for binding or other activity, is then used to find the library with an active peptide. Then twenty new libraries are synthesized and assayed to determine the effective amino acid in the third position, and the process is reiterated in this fashion until the active hexapeptide is defined. This is analogous to the method used in searching a dictionary; the peptide is decoded by construction using a series of sieves or buckets and this makes the search logarithmic.
Large libraries of wholly or partially synthetic antibody combining sites, or paratopes, have been constructed utilizing filamentous phage display vectors, referred to as phagemids, yielding large libraries of monoclonal antibodies having diverse and novel immunospecificities. The technology uses a filamentous phage coat protein membrane anchor domain as a means for linking gene-product and gene during the assembly stage of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial libraries. Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991). Combinatorial libraries of antibodies have been produced using both the cpVIII membrane anchor (Kang et al., supra) and the cpIII membrane anchor. Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991).
The diversity of a filamentous phage-based combinatorial antibody library can be increased by shuffling of the heavy and light chain genes (Kang et al., Proc. Natl. Acad. Sci., USA, 88:11120-11123, 1991), by altering the CDR3 regions of the cloned heavy chain genes of the library (Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-4461, 1992), and by introducing random mutations into the library by error-prone polymerase chain reactions (PCR). Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580, 1992).
Mutagenesis of proteins has been utilized to alter the function, and in some cases the binding specificity, of a protein. Typically, the mutagenesis is site-directed, and therefore laborious depending on the systematic choice of mutation to induce in the protein. See, for example Corey et al., J. Amer. Chem. Soc., 114:1784-1790 (1992), in which rat trypsins were modified by site-directed mutagenesis. Partial randomization of selected codons in the thymidine kinase (TK) gene was used as a mutagenesis procedure to develop variant TK proteins. Munir et al., J. Biol. Chem., 267:6584-6589 (1992).
Using the random synthetic hexapeptide library displayed on filamentous phage, O'Neil et al., Science, 249:774-778 (1990), described the identification of a variety of different hexapeptides that contain the sequence Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD) and that bind to the integrin GPIIb/IIIa.
In another approach, Roberts et al., Gene, 121:9-15 (1992), describes the point mutation of a protease inhibitor (BPTI) as a fusion protein with gene III of a phagemid, and demonstrated a change in binding specificity such that the mutant binds human neutrophil elastase rather than trypsin. Similarly, Roberts et al., Proc. Natl. Acad. Sci., USA, 89:2429-2433 (1992), produced by mutagenesis a library of phage displaying mutant trypsin inhibitor, and isolated variant enzymes with increased affinity.
Tomiyama described an antibody designated PAC-1 which binds the integrin GPIIb-IIIa and contains the sequence Arg-Tyr-Asp (RYD) in the antibody's third complementarity determining region of the heavy chain. Tomiyama et al., J. Biol. Chem., 267:18085-18092 (1992). Antibody PAC-1 is a marker for platelet activation and its binding to GPIIb-IIIa can be blocked using peptides that contain the RGD sequence.